The findings support exploration of clinical treatment regimens that combine potent antiandrogens and PSMA-targeted therapies for prostate cancer.

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The purpose of this study was to elucidate the mechanism of the antihypertensive effect of the angiotensin I (Ang I) converting enzyme inhibitor captopril in spontaneously hypertensive rats (SHR). Drinking responses, peripheral vascular reactivity, and angiotensin II (Ang II) receptor binding in both the brain and vascular smooth muscle were examined in control and captopril-treated SHR. Pregnant and nursing dams were treated with oral captopril (100 mg/kg). After weaning, offspring were maintained on captopril (50 mg/kg). The average systolic pressures after 21 weeks of captopril treatment were 122 +/- 3 mm Hg (male) and 118 +/- 4 mm Hg (female) as compared with 169 +/- 4 mm Hg (male) and 162 +/- 2 mm Hg (female) in age-matched controls. Drinking responses to intracerebroventricular (10 ng) and subcutaneous (100 micrograms/kg) administration of Ang I and II were attenuated in captopril-treated SHR in comparison to control SHR. Ang II receptor binding in the hypothalamus, thalamus, and septum of captopril-treated SHR was also significantly reduced. In contrast to a depressed angiotensinergic system in the brain, peripheral vascular reactivity to Ang II, as determined in isolated, artificially perfused kidneys, was elevated. Threshold and ED50 values for Ang II were significantly lower in captopril-treated SHR than in controls. Ang II receptor binding in aortic smooth muscle cells prepared from captopril-treated SHR was also significantly greater than in cells from controls. Thus, lifetime treatment with captopril induced alterations in the renin angiotensin systems in the periphery and brain that were manifested by changes in receptor binding and responsiveness to Ang II.(ABSTRACT TRUNCATED AT 250 WORDS)

We layered fresh, unprocessed plasma from healthy rats with early (less than or equal to 7 days) or benign, chronic (greater than 3 wk) one-kidney, one-clip hypertension and from paired one-kidney normotensive control rats over confluent primary-cultured rat aortic smooth muscle cells. Plasma from all rats increased cellular ouabain-sensitive 86Rb+ uptake and sodium content and decreased ouabain-insensitive 86Rb+ uptake compared with uptakes and content in the presence of balanced salt solution (P less than 0.01). Cells incubated in the presence of plasma from rats with early (P less than 0.02) or chronic hypertension (P less than 0.01) had significantly reduced ouabain-sensitive 86Rb+ uptake when compared with cells incubated in normotensive plasma, but their intracellular Na+ contents were not lower. We no longer detected this uptake difference when chronic hypertensives drank 0.9% NaCl instead of water. Plasma from hypertensive rats also altered ouabain-insensitive 86Rb+ uptake by the cultured cells. These findings of this new, reproducible, and specific assay system support the hypothesis that plasma factors inhibit the membrane sodium-potassium pump in vascular smooth muscle cells in this form of hypertension. The abnormality occurs in both early and chronic stages, but may not be related to sodium intake. The data also provide evidence for plasma factors in hypertension altering membrane K+ permeability.

Transport and motility inhibitors have been used to classify different types of high-affinity cytochalasin B (CB) binding sites in 3T3 cells. The potency of phloretin and phlorizin as inhibitors of sugar uptake paralleled their effectiveness in displacing high-affinity bound CB from the cells, indicating that the two compounds compete with CB for binding to sites associated with sugar transport proteins. On the other hand, cytochalasins D and E, which did not inhibit sugar uptake, inhibited binding of CB to a portion of the high-affinity sites, most probably those associated with actin-containing cytoskeletal-contractile structures. A small amount of high-affinity CB binding remained in the presence of both phloretin and cytochalasin E, indicating that the cells have a third class of sites which is not related to either sugar transport or cell motility, When isolated membranes were examined, it was found that a fraction of each class of high-affinity CB binding sites were associated with the fraction. In contrast, only sites sensitive to cytochalasin D were recovered in a soluble extract of the cells.

In populations of cultured arterial endothelial and smooth muscle cells grown under the same conditions, we have measured the total activity per cell of 10 enzymes commonly used as "markers" for subcellular organelles: NADH: ferricyanide reductase, NADH:cytochrome c reductase (rotenone insensitive), NADPH:cytochrome c reductase, a-glucosidase, 5'-nucleotidase, alkaline phosphodiesterase I, cytochrome oxidase, monoamine oxidase, cathepsin D, and N-acetyl--glucosaminidase. Significant differences between the cell types were found for 7 of the 10 enzymes tested. The total activity of 5'-nucleotidase in cultured smooth muscle cells was 17 times that of cultured endothelial cells. Comparison of the activities in the two cell types freshly collected and in culture showed that the difference in 5'-nucleotidase in cultured cells is due principally to loss of activity from endothelial cells, suggesting that this activity is regulated differently in the two cell types. In both cell types cathepsin D activity rose during culture. The endothelial cell layer that lines arteries is directly exposed to blood components. The underlying smooth muscle cells are normally exposed only to those components that successfully traverse the endothelial layer, or to compounds synthesized or modified by the endothelial layer. The endothelial layer acts as a selective barrier between smooth muscle cells and blood components, in a manner analogous to the function of a cellular membrane. Benditt and Benditt (1) have shown that proliferating smooth muscle cells in atherosclerotic lesions are monoclonal and have proposed that the lesion is a response to mutagenic agents carried in the blood. Exposure of smooth muscle cells to serum components stimulated proliferation both of cultured cells (2, 3) and apparently of smooth muscle cells in artery when the endothelial layer was damaged (4, 5). This event is a central feature of the model proposed by Ross and Glomset (4) for the development of atherosclerotic lesions. Consideration of each of these models emphasizes the need to better understand the differing responses and interrelation of endothelial and smooth muscle cells.The recent development of techniques for isolating and maintaining pure cultures of endothelial cells (for a recent review, see ref. 6), coupled with established procedures for cultivating smooth muscle cells from the same source, has provided the opportunity to directly compare the biochemical characteristics of these two cell types. We have established smooth muscle and endothelial cells in culture from hog aorta, and have measured the activities of 10 enzymes commonly used as subcellular markers. Significant differences were found between the cell types in their content of a number of these enzymes, most prominently in 5'-nucleotidase, an enzyme whose reaction product is vasoactive. MATERIALS AND METHODSCell Culture. Tissue culture medium for both cell types consisted of Dulbecco-Vogt modified Eagle's medium with additions as described by Bierman et al. (7), with the ...

e16007 Background: Recent approvals of four new prostate cancer (PCa) drugs and a growing number of pipeline agents have created opportunities for designing rational drug combinations. Potent antiandrogens such as enzalutamide and abiraterone affect expression of a host of androgen-regulated molecules, including those that represent targets for therapy. One such target is PSMA, a well-characterized cell-surface antigen abundant on prostate cancer cells. PSMA ADC is a PSMA-targeted antibody-drug conjugate currently in phase II clinical testing, comprised of a fully human IgG1 mAb conjugated to vcMMAE (valine-citrulline monomethylauristatin E). Here we examined the kinetics and reversibility of PSMA induction by potent antiandrogens and their associated effects on the preclinical activity of PSMA ADC. Methods: Androgen-dependent and -independent PCa cell lines with varying basal levels of PSMA expression were cultured for up to one month in the presence of enzalutamide or abiraterone, followed by drug washout. Cells were tested for PSMA expression over time and for susceptibility to cytotoxicity by PSMA ADC. Potential drug synergy or antagonism was evaluated using the combination index method. Results: Enzalutamide (1 mM) increased PSMA expression by approximately 2.5-fold in LNCaP (androgen-dependent) and C4-2 (androgen-independent) cells, with maximal expression observed after approximately 4 weeks’ culture. PSMA expression returned to basal levels within days following removal of enzalutamide from the culture. Enzalutamide and PSMA ADC exhibited synergistic antitumor activity in LNCaP and C4-2 cells (P < 0.05). Similar results were observed for abiraterone in C4-2 cells. Less induction of PSMA expression by antiandrogens and modest effects on cytotoxicity were observed using 22Rv1 cells, an androgen-independent cell line with low basal expression of PSMA. Conclusions: Enzalutamide and abiraterone significantly and reversibly augmented PSMA expression and potentiated the activity of PSMA ADC in PCa cell lines in vitro. The findings support clinical exploration of regimens that combine potent antiandrogens and PSMA-targeted therapies.

A number of proteins that bind specifically to the barbed ends of actin filaments in a cytochalasin-like manner have been purified to various degrees from a variety of muscle and non-muscle cells and tissues. Preliminary evidence also indicates that proteins that interact with the pointed ends of filaments are present in skeletal muscle. Because of their ability to cap one or the other end of an actin filament, we have designated this class of proteins as the 'capactins'. On the basis of their effect on actin filament assembly and interaction in vitro, we propose that the capactins play important roles in cellular regulation of actin-based cytoskeletal and contractile functions. Our finding that the disappearance of actin filament bundles in virally transformed fibroblasts can be correlated with an increase in capactin activity in the extracts of these cells is consistent with this hypothesis.